PROJECT SUMMARY Over the past 10 years it has become increasingly apparent that there is exceptional complexity in the RNA population in cells. This diverse RNA pool includes microRNAs, Piwi-interacting RNAs, termini- associated RNAs and other noncoding RNAs. In many cases, alterations in these RNAs, or proteins that bind to these RNAs, have been linked to a wide range of medical disorders. A major challenge of molecular biology is to determine the function of these fascinating and novel RNA species. In order to understand how these RNAs function in cells, it is highly useful to image the localization and intracellular movements of these RNAs in living cells under a variety of experimental stimuli. We have developed a novel genetically encodable system to fluorescently tag RNAs in cells. This system utilizes an RNA sequence element, termed Spinach, which is appended to an RNA of interest, and switches on the fluorescence of an otherwise non-florescent dye. This dye is based on the structure of the fluorophore in GFP, making Spinach an RNA mimic of GFP. In the Phase I portion of this project we markedly increased the brightness of Spinach in cells, and expanded the use of Spinach to novel noncoding RNAs. We also developed novel expression plasmids for expressing Spinach- tagged RNAs. In this Phase II proposal, we will develop novel concatamerization strategies to label RNAs with tandem repeats of Spinach, thereby increasing the sensitivity of this labeling approach. We also will tag a set of major noncoding RNAs that label cellular structures, in order to provide a suite of plasmids expressing pre-labeled RNAs that are ready to use for researchers. We also will expand the spectral imaging capacity of this approach by developing new fluorophores for Spinach, and new RNA-fluorophore complexes, which will provide the spectral versatility in biologic imaging that is needed by the research community. Based on the past history of successful commercialization of GFP expression systems, we expect that this expression system will have high commercial potential and will accelerate the pace of RNA research.